the p53-p21-dream-cde/chr pathway regulates g2/m cell cycle

164–174 Nucleic Acids Research, 2016, Vol. 44, No. 1 Published online 17 September 2015doi: 10.1093/nar/gkv927

The p53-p21-DREAM-CDE/CHR pathway regulatesG2/M cell cycle genesMartin Fischer1, Marianne Quaas1, Lydia Steiner2,3 and Kurt Engeland1,*

1Molecular Oncology, Medical School, University of Leipzig, Leipzig, Germany, 2Centre for Complexity & CollectiveComputation, Wisconsin Institute for Discovery, Madison, WI, USA and 3Computational EvoDevo Group &Bioinformatics Group, Department of Computer Science, and Interdisciplinary Centre for Bioinformatics, University ofLeipzig, Leipzig, Germany

Received May 18, 2015; Revised September 06, 2015; Accepted September 08, 2015

ABSTRACT

The tumor suppressor p53 functions predominantlyas a transcription factor by activating and downreg-ulating gene expression, leading to cell cycle arrestor apoptosis. p53 was shown to indirectly represstranscription of the CCNB2, KIF23 and PLK4 cell cy-cle genes through the recently discovered p53-p21-DREAM-CDE/CHR pathway. However, it remained un-clear whether this pathway is commonly used. Here,we identify genes regulated by p53 through thispathway in a genome-wide computational approach.The bioinformatic analysis is based on genome-wideDREAM complex binding data, p53-depedent mRNAexpression data and a genome-wide definition ofphylogenetically conserved CHR promoter elements.We find 210 target genes that are expected to beregulated by the p53-p21-DREAM-CDE/CHR path-way. The target gene list was verified by detailedanalysis of p53-dependent repression of the cell cy-cle genes B-MYB (MYBL2), BUB1, CCNA2, CCNB1,CHEK2, MELK, POLD1, RAD18 and RAD54L. Most ofthe 210 target genes are essential regulators of G2

phase and mitosis. Thus, downregulation of thesegenes through the p53-p21-DREAM-CDE/CHR path-way appears to be a principal mechanism for G2/Mcell cycle arrest by p53.

INTRODUCTION

The key functions of the tumor suppressor p53 are regula-tion of the cell cycle and induction of apoptosis. Its dom-inant role is to serve as a transcription factor by activat-ing or downregulating expression of target genes (1,2). Thegene of the cyclin-dependent kinase (CDK) inhibitor p21(WAF1, CIP1, CDKN1A) was the first p53 transcriptionaltarget to be identified and shown to be activated by p53 (3).

Additionally, p21 was later implicated in mediating indirecttranscriptional repression by p53 (4–12). A possible mech-anism for indirect transcriptional repression is based on thecdk-inhibitor function of p21 which leads to hypophospho-rylation of retinoblastoma tumor suppressor pRB-relatedpocket proteins. Low phosphorylation levels of the pRB-related p130 and p107 proteins then induce formation ofa protein complex named DREAM (DP, RB-like, E2F4and MuvB). DREAM assembles as a result of changingcomponents of the MMB (MYB-MuvB) complex (13–15).In contrast to the activating MMB complex, DREAM isa transcriptional repressor (15–18). The p53-p21-DREAMpathway was shown to function through the cell cycle-dependent element (CDE) and cell cycle genes homologyregion (CHR) promoter sites (19). The switch in proteinbinding to the CDE and CHR elements was suggested tocontrol transcriptional downregulation by p53 upon acti-vation of the p53-p21-DREAM-CDE/CHR pathway (19).

Genes regulated by CDE and CHR sites are categorizedas class I and class II genes. Both classes contain func-tional CHR elements, while class I also employs a CDE fournucleotides upstream of a CHR (20). In contrast to CDEsites, CHR elements conform to a well-defined consensus(21). Many promoters of late cell cycle genes contain func-tional CHR elements, but do not require CDE sites (15,21).The requirement for CHR elements stems from the bindingof the MuvB complex, which forms the core of DREAMand MMB, to CHR sites (15,21). Therefore, it is reason-able to categorize genes by their CHR (15,20,21). Whilebound by DREAM, they mediate transcriptional repres-sion in G0 and G1. Later during the cell cycle, DREAM-mediated repression is lost and the CHR contributes to ac-tivation of the previously repressed genes by binding MuvBcomplexes which recruit B-MYB (MMB) and FOXM1 pro-teins (15,21,22).

Previously, three target genes regulated by the p53-p21-DREAM-CDE/CHR pathway were known, namelyCCNB2, KIF23 and PLK4 (19,23,24). With the small num-

*To whom correspondence should be addressed. Tel: +49 341 9725900; Fax: +49 341 9723475; Email: [email protected] address: Martin Fischer, Department of Medical Oncology, Dana–Farber Cancer Institute and Department of Medicine, Brigham and Women’s Hospital,and Harvard Medical School, Boston, MA, USA.

C© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), whichpermits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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ber of established targets, the general importance of the p53-p21-DREAM-CDE/CHR pathway remained to be estab-lished. Thus, we took a genome-wide approach with the aimto identify possibly all genes targeted by this pathway.

The CHR elements of CCNB2, KIF23 and PLK4 displaythe canonical sequence TTTGAA or its functional inversesite TTCAAA (19,23,24). Here, we included a recent compi-lation of CHR sites deviating from the canonical TTTGAA(21). A total of ten CHR variants are known to date withexamples represented by the cell cycle genes Ccna2 (CHRsequence: CTTGAA) (25), Ccnb1 (TTTAAA) (26), Ccnb2(TTTGAA) (15), B-Myb (Mybl2; TAGGAA) (27), Bub1(TTCGAA), Chek2 (TTTGTA), Melk (TTTGAT), Pold1(TTTGAG), Rad18 (TTCGAG) and Rad54l (TTCGAT)(21). Furthermore, we employed genome-wide chromatinimmunoprecipitation (ChIP) data on DREAM binding (13)and genome-wide p53-dependent gene expression data froma meta-analysis (28). Our results provide a global map of210 genes that are most likely regulated by the p53-p21-DREAM-CDE/CHR pathway. With many of these genesperforming essential functions in G2 phase and mitosis,the results suggest that the p53-p21-DREAM-CDE/CHRpathway represents a key mechanism in p53-mediated cellcycle arrest.

MATERIALS AND METHODS

Computational analyses

Sources of primary data for the analyses were six studies onp53-dependent RNA expression (29–34) and ChIP resultson binding of DREAM components E2F4, LIN9, LIN54and p130 (13). A previously published list of 19 736 knownprotein-coding genes including their identifiers, p53 Expres-sion Score and DREAM binding information served as abasis for the analyses (28). Furthermore, potential pathwaytarget genes were selected for CHR sites annotated in thepromoter regions of the genes. String representation for theCHR was chosen as follows: TTTGAA, TTTAAA, TTC-GAA, TTTGTA, CTTGAA, TTTGAG, TTTGAT, TTC-GAG, TTCGAT and TAGGAA (21). CHR elements weresearched in the region of 1000 bp upstream and downstreamfrom the transcriptional start site (TSS) on both strandsthat were not extended into the coding sequence or othergenes located upstream of the TSS. PhastCons conservationscores (35) obtained from the multiz46 alignment of pla-cental mammalia (36) were used to calculate average phylo-genetic sequence conservation. Only those hits were listedthat have an average PhastCons conservation score of atleast 0.9. Results are displayed in Supplementary Table S2.Genes that are bound by DREAM, possess a conservedCHR and display a p53 Expression Score ≤ −3 are pre-sented as strong candidate targets of the p53-p21-DREAM-CDE/CHR pathway in Table 1. Pathway enrichment anal-ysis was carried out using the DAVID Functional Annota-tion tool (37).

Cell culture and drug treatment

HCT116 wild-type and HCT116 p21−/− cells (38), HFF,and NIH3T3 cells (DSMZ, Braunschweig, Germany) weregrown in Dulbecco’s modified Eagle’s medium (DMEM;

Lonza, Basel, Switzerland) supplemented with 10% fe-tal calf serum (FCS) (Biochrom, Berlin, Germany) andpenicillin/streptomycin and maintained at 37◦C and 10%CO2. Cells were treated with DMSO (15 �l), doxorubicin(0.2 �g/ml; Medac, Wedel, Germany), nutlin-3a (10 �M;Cayman Chemicals, Ann Arbor, MI, USA) or 5-FU (25�g/ml; Sigma, Taufkirchen, Germany) as indicated.

Flow cytometry

Cells were fixed for at least 12 h at 4◦C in one volumephosphate-buffered saline/1 mM EDTA and three volumesof absolute ethanol. DNA was stained with propidium io-dide at a final concentration of 10 �g/ml in presence ofRNase A (10 �g/ml). DNA content per cell was measuredby flow cytometry on an LSR II instrument (Becton Dick-inson, Franklin Lakes, NJ, USA). Cell sorting was carriedout on a FACSVantage SE (Becton Dickinson). Data anal-ysis was carried out with WinMDI 2.9 software.

RNA extraction, reverse transcription and semi-quantitativereal-time polymerase chain reaction (PCR)

Total cellular RNA was isolated using TRIzol Reagent (In-vitrogen, Carlsbad, CA, USA) following the manufacturer’sprotocol. One-step reverse transcription and quantitativereal-time PCR were performed with an ABI 7300 Real-Time PCR System (Applied Biosystems, Forster City, CA,USA) using QuantiTect SYBRGreen PCR Kit (Qiagen,Hilden, Germany) as described previously (19,24). Primersequences are listed in Supplementary Table S1.

Chromatin immunoprecipitation (ChIP)

HCT116 and HFF cells were cross-linked with 1%formaldehyde for 10 min at room temperature. ChIP wasperformed as described previously (19,24). The follow-ing antibodies were used for precipitation of transcrip-tion factors: E2F4 (C-20, Santa Cruz Biotech.), p130 (C-20, Santa Cruz Biotech.), p53 (Ab-6, DO-1, Calbiochem),LIN9 (ab62329, Abcam, Cambridge, UK). A second LIN9antibody was a kind gift from James DeCaprio (13). A non-targeting IgG polyclonal rabbit antibody was used as a con-trol for non-specific signals. For all precipitations 1–2 �g ofantibody and 20–35 �l of Protein G Dynabead suspension(Invitrogen) were used. Immunoprecipitated DNA was usedas template for quantitative real-time PCR as described pre-viously (19,24). Primer sequences are listed in Supplemen-tary Table S1.

RESULTS

The p53-p21-DREAM-CDE/CHR signaling pathway regu-lates 210 potential target genes

Three sets of information were utilized to identify possiblyall genes regulated by the p53-p21-DREAM-CDE/CHRpathway: Genome-wide RNA expression dependent onp53, ChIP data on binding of DREAM components in thehuman genome, and genome-wide detection of phylogenet-ically conserved CHR elements.


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Table 1. The p53-p21-DREAM-CDE/CHR signaling pathway regulates 210 potential target genes

ADSS CDC25C DLEU1 IFT80 MND1 RAD54LANLN CDC7 DLGAP5 INCENP MTF2 RANGAP1ANP32E CDCA2 ESCO2 ING1 MYBL2 RBM15ARHGAP11A CDCA3 ESPL1 ING3 NASP RBMXARHGAP11B CDCA5 EXOSC8 IQGAP3 NCAPD2 REEP4ARL13B CDCA8 EXOSC9 KIAA1731 NCAPD3 RIF1ARL6IP1 CDK1 FAM64A KIF11 NCAPG RNASEH2AASF1B CDK2 FAM83D KIF14 NCAPG2 RTKN2ASPM CDKN3 FANCB KIF15 NCAPH SASS6ATAD2 CENPA FBXO5 KIF18A NDC80 SCLT1AURKA CENPE FOXM1 KIF20A NEIL3 SGOL1AURKB CENPF GAS2L3 KIF20B NET1 SGOL2BIRC5 CENPL GSG2 KIF22 NUF2 SHCBP1BORA CENPM GTSE1 KIF23 NUP107 SLC25A40BUB1 CENPN H2AFX KIF24 NUP205 SMC2BUB1B CENPO H2AFZ KIF2C NUP35 SMC4C11orf82 CEP152 HAUS8 KIF4A NUSAP1 SNRPAC12orf32 CEP55 HIST1H2AE KIFC1 OIP5 SP4C15orf42 CHEK2 HIST1H2AM KPNB1 ORC1 SPAG5C2orf69 CIT HIST1H2BF LIN54 PCNT SPC25C3orf26 CKAP2L HIST1H2BH LIN9 PLK1 STILC9orf100 CKAP5 HIST1H2BI LMNB1 PLK4 SUZ12CACYBP CKS1B HIST1H2BM LRRC49 POC5 TCERG1CASC5 CKS2 HIST1H2BN LSM5 POLD1 TMEM48CBX3 CSE1L HIST1H3C MAD2L1 POLQ TMPOCCDC150 CSTF1 HIST1H3D MASTL POP7 TPX2CCDC18 CTDSPL2 HIST1H4C MCM5 PPIH TRAIPCCDC34 DARS2 HIST2H2AB MCM7 PRC1 TROAPCCDC99 DBF4B HIST2H2AC MCM8 PRIM2 TTKCCNA2 DCAF16 HJURP MDC1 PRPF38A UBE2CCCNB1 DCK HMGB2 MELK PRR11 UBE2SCCNB2 DCLRE1B HMMR METTL13 PTTG1 UNGCDC20 DDX10 HNRNPA0 METTL4 RACGAP1 USP1CDC25A DEPDC1 HNRNPA2B1 MIS18BP1 RAD18 YEATS4CDC25B DEPDC1B HNRNPUL1 MKI67 RAD21 ZNF367

Data from a meta-analysis of six genome-wide p53-dependent gene expression analyses to identify genes thatare repressed by p53 formed one basis for the analysis (28–34). In each study, a gene could be identified as activated(positive score; +1) or repressed (negative score; −1) by p53.By calculating the sum over all analyses, p53-dependent Ex-pression Scores ranging from −6 to +6 were assigned, rep-resenting direction as well as reproducibility of regulation(28). An Expression Score ≤ −3 was a criterion for a geneto be considered as repressed by p53. As a second dataset, binding results of DREAM components E2F4, LIN9,LIN54 and p130 from ChIP analyses were employed to de-tect DREAM target genes (13). A gene was scored positivefor DREAM binding if three of the four components weredetected. The third selection criterion was a conserved CHRsite in the promoter of a gene. CDE sites were not used be-cause many potential target genes were expected to employsolely a CHR and not require a CDE (20). Since all CDEsites require CHR elements for their function, focusing thesearch on CHR sites covers all CHR- and CDE/CHR-regulated genes (20,21). CCNB2, KIF23 and PLK4, thethree genes thus far shown to be regulated by the p53-p21-DREAM-CDE/CHR pathway, bind DREAM through thecanonical CHR sequence TTTGAA (19,23,24). Recently,variants of CHR sites were identified in a comprehen-sive genome-wide screen. This yielded a list of ten func-tional CHR variants, namely TTTGAA, TTTAAA, TTC-GAA, TTTGTA, CTTGAA, TTTGAG, TTTGAT, TTC-GAG, TTCGAT and TAGGAA (21). Here, we employed

all of these CHR motifs in a genome-wide search to obtainan essentially complete set of genes regulated via CHR- andCDE/CHR sites. In order to score positive as a CHR site,phylogenetic conservation of potential CHRs within 1000bp upstream or downstream of annotated transcriptionalstart sites (TSS) was required.

We found 870 genes bound by DREAM in proximity totheir TSS (Supplementary Table S2). Out of these 870 genes,358 (41.1%) were repressed by p53 displaying an ExpressionScore of ≤ −3. When all three selection criteria were appliedwith binding of three out of four DREAM components, re-pression by p53 with an Expression Score ≤ −3, and pres-ence of one of the ten CHR variants being phylogeneticallyconserved in the promoter, we identified 210 genes as targetsfor the p53-p21-DREAM-CDE/CHR pathway (Figure 1;Table 1, Supplementary Table S2). The three known targetsof the pathway, CCNB2, KIF23 and PLK4, were detectedby the analysis. This demonstrates the ability of this screen-ing approach to identify bona fide candidates. GAS2L3 hadbeen missed by the search because only two of the DREAMcomponents were above the threshold (Supplementary Ta-ble S2), although it had been described as a DREAM tar-get (39). Also, B-MYB (MYBL2) was previously shown tobe a CHR-controlled gene downregulated by p53 (21,28).Therefore, GAS2L3 and MYBL2 were included in the list ofgenes regulated by the p53-p21-DREAM-CDE/CHR path-way (Table 1).



Figure 1. Genes associated with cell cycle and mitosis are enriched among targets of the p53-p21-DREAM-CDE/CHR pathway. Venn diagram displayingthe overlap in the groups of genes found as repressed by p53, bound by DREAM, and containing a conserved CHR element. Top 10 GO terms enrichedamong the 210 genes displayed in Table 1 and the other group overlaps as identified using the DAVID Functional Annotation tool (Supplementary TableS3).

Repression of non-canonical CHR-containing genes by p53requires p21 and is conserved between mouse and human

With CCNB2, KIF23 and PLK4, only genes containingcanonical CHR sites had been tested in detail for their reg-ulation through the p53-p21-DREAM-CDE/CHR path-way. In order to examine several of the new targets iden-tified through the bioinformatic analysis for their regu-lation on a single-gene basis, we chose candidate geneswhich mostly contain CHR sites deviating from the canon-ical TTTGAA. We investigated p53-dependent regulationof B-MYB (MYBL2), BUB1, CCNA2, CCNB1, CHEK2,MELK, POLD1, RAD18 and RAD54L, which hold repre-sentative non-canonical CHR motifs that have been shownto bind DREAM (21). Relative mRNA expression wasdetermined in untreated NIH3T3 cells in comparison tocells treated with the solvent DMSO, the MDM2 in-hibitor nutlin-3a or the pyrimidine analogue 5-FU. We usedNIH3T3 cells because cell cycle-dependent regulation ofthese genes was demonstrated in this cell line in a previ-ous study (21) and results from this mouse cell line wouldcomplement the data from the human cell systems (Supple-mentary Table S2). Indeed, we found all genes with non-canonical CHR elements to be downregulated upon p53activation (Figure 2A, Supplementary Figure S1). Next,we investigated p53-dependent regulation of the human

gene orthologs in HCT116 wild-type, HCT116 p21-negativeand non-cancerous HFF cells (Figure 2B–D, Supplemen-tary Figure S1). Again, we found all genes containing anon-canonical CHR to be repressed following p53 activa-tion in HCT116 and HFF cells, similar to the observa-tions in NIH3T3 cells. These results support the data fromour genome-wide screening and provide evidence that p53-dependent repression of these genes is conserved betweenmouse and human. Importantly, p53-dependent repressionis essentially lost in HCT116 p21−/− cells (Figure 2C). Inagreement with our data, p21-dependent downregulationhas been reported for four of the nine genes tested here,namely CCNA2 (4,6,11,40), CCNB1 (6–8,11), POLD1 (11)and RAD54L (7). These results are consistent with a mecha-nism involving indirect, p21-mediated repression upon p53stabilization. Based on these findings, we conclude that p53-dependent repression of these CHR-containing genes re-quires p21.

p53 does not bind to promoters of genes harboring CHR sites

The finding that all genes tested require p21 for p53-dependent repression contrasts the previously proposed di-rect repression by p53 for two of the genes, CCNB1 andPOLD1 (41–44). Thus, we tested for p53 binding to thepromoters of CCNB1 and POLD1 as well as B-MYB



Figure 2. Repression of non-canonical CHR-containing genes by p53 requires p21 and is conserved between mouse and human. The log2-fold change ofmRNA expression from treated compared to untreated (A) NIH3T3, (B) HCT116 wild-type, (C) HCT116 p21−/− and (D) HFF cells is displayed. Cellswere treated with doxorubicin, nutlin-3a or 5-FU for 24 h. Untreated cells and cells treated with DMSO served as controls. Normalization was carried outagainst measurements from untreated cells. GAPDH, L7 and U6 served as negative controls for p53 response, while CDKN1A and MDM2 were employedas positive controls. (A–C) Experiments were performed with two biological replicates and three technical replicates each (n = 6). (D) Experiments wereperformed with three technical replicates (n = 3). Significance of changes in expression levels was tested against GAPDH expression levels using the unpairedStudent’s t-test; n.s. not significant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.



(MYBL2), BUB1, CCNA2, CHEK2, MELK, RAD18 andRAD54L in HCT116 wild-type, HCT116 p21−/−, or HFFcells, untreated or treated with doxorubicin (Figure 3).Binding of p53 to promoters with CHR sites was not ob-served. These findings do not support a mechanism of di-rect repression by p53 (41–44), but are consistent with theindirect p53-p21-DREAM-CDE/CHR pathway which re-quires p21 (Figure 2).

p53 induces p21-dependent binding of DREAM to genes withCHR sites

The CHR sites in the B-MYB (MYBL2), BUB1, CCNA2,CCNB1, CHEK2, MELK, POLD1, RAD18 and RAD54Lgenes have previously been shown by DNA affinity purifica-tion assays to bind DREAM in vitro (21). Here, we tested fordifferential DREAM binding by ChIP in cells before andafter p53 activation. Binding of the DREAM componentsLIN9, E2F4 and p130 to the promoters of these genes wereexamined upon p53 induction by doxorubicin. Binding ofthe MuvB core component LIN9 was found to be enrichedat some but not all promoters (Figure 4A). In contrast, theDREAM components E2F4 and p130 showed increasedbinding at all CHR-containing promoters in HCT116 wild-type cells treated with doxorubicin compared to untreatedcells (Figure 4A). These findings are consistent with themodel developed from the regulation of CCNB2, KIF23and PLK4, which contain canonical CHR sites (19,23,24).Moreover, DREAM binding to the promoters was generallyreduced in HCT116 p21−/− cells and did not increase afterdoxorubicin treatment of HCT116 p21−/− cells (Figure 4B).Interestingly, binding of E2F4 was observed for most genesto be reduced in doxorubicin-treated compared to untreatedHCT116 p21−/− cells (Figure 4B). A likely explanation isthe cell cycle shift toward G2/M upon doxorubicin treat-ment that causes the expected decrease in DREAM stabil-ity when p21 cannot inhibit the CDKs (Figure 4B and C).Thus, the p53-p21 pathway appears to stabilize formationof DREAM and enhances its binding to promoters in wild-type cells despite the doxorubicin-induced G2/M cell cycleshift. Taken together, the observations on these nine individ-ual genes suggest that p21 is required for DREAM bindingto CHR promoters upon p53 activation.

G2/M cell cycle genes are among the targets of the p53-p21-DREAM-CDE/CHR signaling pathway

We performed a Gene Ontology analysis of the 210 targetgenes using the DAVID Functional Annotation tool (37).Cell cycle-associated terms were prominently represented inthis set of genes (Figure 1, Supplementary Table S3). Par-ticularly, genes encoding proteins required in late phases ofthe cell cycle from G2 through mitosis comprised the largestfraction in the analysis. When comparing the 210 genes toan analysis of four genome-wide cell cycle expression stud-ies (21), it was observed that 158 (75.2%) of the CHR genesshow cell cycle-dependent regulation. More precisely, thevast majority of genes regulated by the p53-p21-DREAM-CDE/CHR pathway displayed their peak expression in G2phase or mitosis.

DISCUSSION

In this study, a list of target genes for the p53-p21-DREAM-CDE/CHR signaling pathway is presented. Selection ofthe 210 candidate targets was performed using stringentcriteria to minimize the number of false-positives. Identi-fication of the known p53-p21-DREAM-CDE/CHR tar-gets CCNB2, KIF23 and PLK4 demonstrates the abil-ity of the screening approach to identify bona fide can-didates. These three genes harbor the canonical CHR se-quence TTTGAA in their promoters (19,23,24). Notably,the non-canonical CHR elements TTTAAA, TTCGAA,TTTGTA, CTTGAA, TTTGAG, TTTGAT, TTCGAG,TTCGAT and TAGGAA had not yet been tested for theirfunction in the p53-p21-DREAM-CDE/CHR pathway(21). Thus, B-MYB (MYBL2), BUB1, CCNA2, CCNB1,CHEK2, MELK, POLD1, RAD18 and RAD54L as rep-resentative genes harboring the nine non-canonical CHRvariants were tested for their response to p53 activation.Detailed experiments with all of these genes confirmed re-sults from the genome-wide analyses and supported the no-tion that the genes are regulated by this pathway (Figures2–4). DREAM, represented by its components p130 andE2F4, was shown to be recruited to their promoters (Fig-ure 4) (19). DREAM binding to the specific CHR sites hasalso been established (21). In agreement with our observa-tions, it has been reported that p53-dependent downregu-lation of the mouse homologs Bub1, Ccna2 and Ccnb1 re-quires p107 and p130 (45). These pRB-related pocket pro-teins were later identified as components of the DREAMcomplex (13,14,18). This leads to the conclusion that thep53-p21-DREAM-CDE/CHR pathway functions throughall known CHR variants and is largely conserved betweenmouse and human.

Furthermore, there are two indicators that threshold set-tings for the genome-wide analysis were adjusted to yieldhigh-confidence candidates. One indicator is that all can-didates tested in detail for their regulation by the path-way were confirmed (Figures 2–4). This suggests that therate of false-positives may be low. The other observationis that GAS2L3 was not selected as a pathway gene. Sincethe DREAM binding threshold required three of the fourDREAM components tested to be detected in the ChIPscreen. However, in the case of GAS2L3 only two compo-nents were found positive for binding in the screen (Sup-plementary Table S2) (13), although GAS2L3 had been re-ported to be a DREAM target (39). Taken together, theseobservations indicate that threshold settings were so strin-gent that the computational analysis rather missed candi-dates than to include false-positive genes. This suggests thatthe 210 genes in Table 1 are indeed strong candidate tar-gets of the p53-p21-DREAM-CDE/CHR pathway but thatsome additional target genes may have been missed.

GO term enrichment analysis of the 203 genes that con-tain a conserved CHR and are repressed by p53, but do notbind DREAM, shows a small enrichment also for cell cy-cle genes (Figure 1). Considering that GAS2L3 belonged tothat group before we manually assigned it as DREAM tar-get, it is likely that more currently unknown DREAM targetgenes can be found among this group of genes. The group of200 genes binding DREAM, possessing a conserved CHR,



Figure 3. p53 does not bind to promoters of genes harboring CHR sites. Protein binding to promoters of the indicated genes was tested by ChIP in (A)HCT116 wild-type cells, (B) HCT116 p21−/− and (C) HFF cells either left untreated or treated with doxorubicin for 24 h (HFF) or 48 h (HCT116) followedby real-time PCR. The CDKN1A promoter served as a positive control for p53 binding; the GAPDHS promoter which does not bind p53 was used as anegative control. One representative experiment with three technical replicates (n = 3) is displayed.



Figure 4. p53 induces p21-dependent binding of DREAM to genes with CHR sites. Protein binding to CHR-containing promoters in untreated or 48 hdoxorubicin-treated HCT116 (A) wild-type or (B) p21−/− cells was tested by ChIP followed by real-time PCR. Protein binding to the GAPDHS promoterserved as negative control. One representative experiment with three technical replicates (n = 3) is displayed. Significance was tested using the pairedStudent’s t-test; n.s. not significant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (C) Flow cytometry of cells used in Figure 4; cells were stained with propidiumiodide (PI).



and that are not found to be repressed by p53 likely con-tains genes that either are repressed by p53 but were misseddue to the high stringency criteria in our analysis or genesthat might be regulated solely by DREAM in the cell cy-cle. Interestingly, GO term analysis of the 150 genes thatbind DREAM and are repressed by p53, but do not pos-sess phylogenetically conserved CHR elements, shows highenrichment for biological processes of early (G1/S) cell cy-cle genes, such as DNA replication and DNA metabolism(Figure 1, Supplementary Table S3).

Cell cycle genes are general targets for p53-mediateddownregulation independent of cell type or method ofp53 activation. In regard to the underlying mechanism, ei-ther no or contradictory details have been reported. Sev-eral cell cycle genes among the 210 targets have been sug-gested to be directly repressed by p53. Examples includeANLN (46), AURKA (29), CDC20 (47), CDC25B (48),CDK1 (CDC2) (49), PRC1 (50) or PTTG1 (51). However,these genes were not found to be directly repressed by p53in several genome-wide studies (52–54). Moreover, recentevidence suggests that p53 does not directly repress tar-get genes (28). These observations are consistent with ourdata, which provide no evidence for direct repression ofCCNB1 and POLD1, although these genes had been re-ported as direct p53 targets (Figures 2–4) (41–44). Addi-tionally, for a large number of cell cycle genes the mech-anism underlying p53-dependent repression remained un-resolved, e. g. CDC25A (55), CKS1B (56), CKS2 (57) orHMMR (RHAMM) (58). However, for several other genesindirect repression via p21 was reported, including BUB1B(59,60), CENPA (61), CENPE (61), CENPF (59), FOXM1(62) and MAD2L1 (59,60). All of these genes can be foundamong the 210 target genes (Table 1). Two previous stud-ies had suggested that many cell cycle genes are repressedby p53 via p21 and E2F4, with the earlier of the two reportsshowing DREAM components and CHR sites as being partof this regulation and the later comparing E2F4 bindingwith p53-dependent expression data (19,63). Here, we tooka genome-wide approach combining p53-dependent expres-sion, binding of several DREAM components and selec-tion of CHR-containing genes. We provide evidence that theE2F4-containing DREAM complex represses many, if notall, of these genes upon p53 activation and that this complexis targeted to many promoters via CDE/CHR motifs. Thus,the present analysis offers a mechanism for p53-dependentrepression of these genes, resolving contradictions betweenearlier reports.

Interestingly, expression of most of the 210 candidategenes varies during the cell cycle. Expression levels peak inthe late cell cycle phases, which correspond to the functionof their encoded proteins in G2 and mitosis. Thus, the re-sults suggest that repression of these cell cycle genes servesas a mechanism for p53 to stop cell division, particularly inG2/M. This mechanism of p53-dependent cell cycle controlis exemplified by downregulation of key cell cycle regula-tors such as the cyclins CCNA2, CCNB1 and CCNB2, thecyclin-dependent kinases CDK1 and CDK2, as well as thekinases and phosphatases AURKA, AURKB, PLK1, PLK4,CHEK2, CDC25A and CDC25C. In addition to fast re-sponses which stop cell cycle progression such as transcrip-tional suppression by cyclin F (64), the p53-p21-DREAM-

CDE/CHR pathway may contribute to a permanent cell cy-cle arrest. In agreement with this model, the DREAM com-plex was shown to be important for permanent proliferationarrest during senescence (65).

Taken together, we establish a target list of the p53-p21-DREAM-CDE/CHR signaling pathway. The results alsosuggest a mechanism for the regulation of cell cycle genesthat are targeted by this pathway. Most of the genes aredominantly expressed in G2 phase and mitosis and essentialfor the progression through the late cell cycle. Thus, down-regulation of these genes through the p53-p21-DREAM-CDE/CHR pathway appears to be a principal mechanismfor G2/M cell cycle arrest by p53.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

ACKNOWLEDGEMENTS

We are indebted to Carola Koschke and Andrea Rothe forexpert technical assistance as well as Kathrin Jager, AndreasLosche and Knut Krohn for performing DNA sequencingand flow cytometry. We thank Bert Vogelstein for the kindgift of HCT116 cell lines, Larissa Litovchick and James De-Caprio for LIN9 antibody and Christine Engeland for crit-ical reading of the manuscript. This work was supportedby a postdoctoral fellowship provided by the Fritz ThyssenFoundation (to M.F.), a graduate fellowship provided bythe Freistaat Sachsen (to M.Q.) and the grant ‘Origins andEvolution of Regulation in Biological Systems’ (Grant ID:24332) by the John Templeton Foundation (to L.S.). Theopinions expressed in this publication are those of the au-thors and do not necessarily reflect the views of the funders.Author Contributions: M.F. conceived and K.E. supervisedthe study. M.F. and M.Q. performed the experiments. L.S.and M.F. performed the computational analyses. M.F. andK.E. wrote the manuscript. All authors read and approvedthe final manuscript.

FUNDING

Fritz Thyssen Foundation [to M.F.]; Freistaat Sachsen [toM.Q.]; John Templeton Foundation [Grant ID 24332 toL.S.]. Funding for open access charge: German ResearchFoundation (DFG) and University of Leipzig within theprogram of Open Access Publishing.Conflict of interest statement. None declared.

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the p53-p21-dream-cde/chr pathway regulates g2/m cell cycle

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